In Gram-negative bacteria N-acyl-L-homoserine lactones (AHLs) are produced as quorum-sensing (QS) signaling molecules to promote multicellularity and virulence. With respect to Pseudomonas aeruginosa, N-3- oxo-dodecanoyl-L-homoserine lactone (C12) and N-butanoyl-L-homoserine lactone are utilized by the bacteria to conduct its QS-controlled activities. In contrast to other pathogenicity-promoting factors produced by P. aeruginosa, C12 has been demonstrated to exert a broad spectrum of biological activities in mammalian cells, including host immunomodulation, and was found to be pathologically relevant in P. aeruginosa infections of cystic fibrosis patients. Our laboratories have recently reported evidence linking the immunosuppressive nature of C12 to its ability to selectively disrupt the regulation of NF-?B functions in macrophages and other cell types activated by TLR agonists, TNF or whole bacteria (P. aeruginosa). Interestingly, despite inhibition of NF-?B-dependent immune system effectors, exposure of mammalian cells to C12 resulted in activation of the endoplasmic reticulum (ER) stress response, protein kinase p38 pathway and apoptosis. Notably, unlike TLR agonists and other pathogen-associated molecular patterns (PAMPs), C12-mediated activation of mammalian cells was shown to occur through mechanism(s) distinct from currently known PAMP recognition receptor pathways. At present, the mechanisms by which mammalian cells are able to respond to C12 in such a paradoxical manner are unknown. In bacteria, C12 exerts its effects through interaction with LuxR-type proteins; however, no obvious LuxR homologue exists in mammalian cells. Thus, identification of mammalian cell C12 receptor proteins should provide great insight into a biochemical understanding of AHL-mediated mechanisms of bacterial immunosuppression and lead to the identification of new members of the pattern recognition receptor family. Moreover, the uncovering of such proteins may lead to the discovery of new therapeutic avenues for preventing and/or combatting infections caused by P. aeruginosa, which is of particular significance, as innovative therapeutic and prophylactic strategies are desperately needed for targeting Gramnegative pathogens. To discover C12 target proteins, we have designed a research program focused on the utilization of synthetic C12 analogues as chemical probes. More specifically, clickable C12-based chemical probes will be employed to identify the mammalian cell receptor(s) of C12 via a click chemistry-based, proteomics-mediated approach. To then validate mammalian cell receptor(s) of C12, a combination of fluorescence microscopy, RNAi and functional biochemical assays, or alternatively, protein overexpression, will be used. From these studies, we hope to validate C12 target proteins and begin to provide functional links between these proteins and the biological activities of C12.